Influenza and flaviviruses cause a variety of infectious diseases for which there is no adequate therapeutic agent. The disadvantages of existing treatments include the onset of clinical resistance within thirty six hours and the ineffectiveness of the agents against influenza B. Killed influenza virus vaccines have been available for over sixty years. However, these vaccines have not lessened the morbidity, mortality or severe financial loss caused by this disease. It follows that an agent which treats or prevents an influenza or flavivirus infection or is effective at preventing the clinical symptoms associated therewith will result in a significant benefit to society.
Currently, the only compounds approved for the therapeutic and prophylactic treatment of influenza infections are the adamantanes: amantadine and rimantadine. These compounds inhibit influenza A by inhibiting the function of the M2 ion channel activity of the virus. Amantadine is a potent in vitro inhibitor of influenza A virus as demonstrated by standard antiviral assays such as the plaque reduction assay. Amantadine is effective in reducing the duration of fever and other systemic complaints including but not limited to myalgia (muscular ache) and fatigue when administered to individuals infected with influenza A within forty-eight hours of the onset of clinical symptoms. It has also been observed that amantadine results in a one hundred-fold decrease of virus titer in the nasal washes of human volunteers infected with wild-type influenza virus which correlates with a dramatic decrease in fever score. Thus, in vitro influenza inhibition is predictive of useful in vivo effects, i.e. a reduction of the clinical symptoms associated with the influenza infection.
The present invention derives from the fact that orthomyxoviruses such as influenza and flavivirues such as bovine diarrheal virus and hepatitis C virus are enveloped viruses in which the virus envelope must be fused with the endosomal membrane of the host cell in order to initiate the process of introducing the virus' genetic information into the cell. Because this process is common to all enveloped viruses, it is an attractive target for antiviral chemotherapy. The fusion domain of the envelope glycoprotein of influenza, hemagglutinin (HA) and the envelope protein (E protein) of flaviviruses have been well characterized. See e.g., White J. M, Annu. Rev. Physiol. vol. 52, pages 675-697 (1990); Rev, F. A. et al. Nature, 375, 291-298 (1995), which is herein incorporated by reference.
Influenza virus HA and flavivirus E proteins provide at least two distinct functions: 1) recognition of the host cell receptor, i.e., sialic acid residues on glycoconjugates, and 2) fusion of the viral envelope with the endosomal membrane. Both functions are essential, for example, in the propagation of influenza virus in vitro and in vivo. During influenza viral maturation, monomeric HA is inserted into a lipid bilayer, post-translationally modified and oligomerized into a trimer of identical subunits (trimeric HA). The infectivity of the progeny influenza virus is contingent upon a site-specific cleavage of HA by host cell protease(s). This cleavage results in the formation of two polypeptide chains, HA1 and HA2, which remain associated by non-covalent interactions as well as by an intermolecular and intramolecular disulfide bonds.
It has been established that influenza HA and E protein have two functionally relevant conformations. One conformation exists as a metastable structure at neutral pH and mediates receptor recognition. Following receptor mediated binding to the host cell, the virus is transported to the endosomal compartment where it encounters an acidic environment. The low pH triggers a dramatic structural rearrangement that results in the formation of the other, more stable conformation. It is this structural rearrangement, for example from Form A to Form B of HA in the case of influenza, that allows the fusion domain of HA to directly interact with the endosomal membrane enabling the release of viral genetic information into the host cell cytoplasm. These considerations lend themselves to the development of a strategy for antiviral intervention based on the abrogation of HA-mediated or E protein-mediated fusion of virus-host membranes.